With the development of life sciences, the relevant biomacromolecule separation has become an extraordinary important task. At present, the separation and purification technology for preparing proteins in large scale has become a limiting factor in the development of biotecimology. In respective countries, a great deal of researches has been carried out on the aspect which has also become one of the emphases in technological investments.
Though the research on bio-separation media has been lasted for tens of years and the media have been greatly improved in the performances (such as, mechanical strength, application scope and service life) and separation ability thereof, it is still hard to harmonize the 3 factors such as resolution, capacity and separation speed. Especially for the biomacromolecules, because of their large volumes and changeful structures, the demand on the separation conditions is higher. However, the general synthesized porous media make use of the phase separation between cross-linked polymers and liquid lean solvents and non-solvents (referred to as diluents or pore-making agents) to form channels, and therefore, such a mechanism will necessarily result in pore diameters in nanometer grade (100-300 Å). As the pore diameters are relatively small, the mass transfer process of the mobile phase in the channels is accomplished mainly depending on molecule diffusion, which results in the slow mass transfer speed and long separation time for the biomacromolecules. In order to overcome this disadvantage, it is demanded imperatively to develop a new pore-forming method to prepare polymeric media with large pore diameters (10 or 20 times larger than the diameters of the biomacromolecules).
The above important demand and enormous market perspective make the research on the preparation and application of super-macroporous separation media draw much attentions in respective countries. The researchers in respective countries have developed the following several pore-making methods for preparing super-macroporous media.
(1) A suspension polymerization method taking polymers as pore-making agent: soluble polymers are used to replace the above liquid pore-making agents, see V. L. Sederel, G. J. De Jong, Styrene-divinylbenzene copolymers, Construction of porosity in styrene divinylbenzene matrices. J. Appl. Polym. Sci., 1973, 17, 9: 2835-2846. With the polymerization of the monomers in the liquid droplets, a phase separation occurs between the newly formed cross-linked polymers and the primary polymers being pore-making agent, and finally, relatively larger pore diameters can be obtained by extracting out the polymers being pore-making agent. However, because the phase separation degree between both polymers is large, it is difficult to form a continuous phase of the pore-making agent and therefore it is hard to obtain penetrated macropores. Additionally, it is also relatively difficult to extract completely the polymers as the pore-making agent by solvents.
(2) A nanoparticle condensation method: The media prepared using this method have been commercialized, which are referred as POROS Perfusion Chromatography media [U.S. Pat. No. 5,019,270, U.S. Pat. No. 5,228,989, U.S. Pat. No. 5,833,861]. At the end of 1980s, the POROS media have been used in the process of column chromatography separation. These materials have two kinds of pores: 6000-8000 Å of through pores and 800-1500 Å of diffusion pores. The liquid in the through pores can flow in a manner of convection, and thus the mass transfer speed thereof is fast; and the diffusion pores provide a sufficiently high specific surface area so as to ensure the adsorption capacity of the media; and additionally, because the distance between the through pores and the diffusion pores is short, the mobile phase can diffuse therein easily. The occurrence of this kind of media brought great inspiration to scientists at that time. However, the preparation of POROS media is relatively difficult: firstly, particles in nanometer grade are prepared, and then these small particles are “stuck” to form clusters and further congregated into particles in micron grade. The channels in the particles are constituted of the apertures among the small particle clusters. It can be seen from the above preparation method that, it is very difficult to control the pore diameters by controlling the irregular congregating state among the nanoparticles, which result in the poor repeatability of batches, low yield of products and expensive price for POROS media. Furthermore, the cohesive force among the nanoparticles is low such that the media is weak in strength, difficult to be loaded into a column, and liable to be broken during the application. Therefore, at present, the application of the POROS media is stagnant and if a new method can overcome the disadvantages thereof, it will bring new vitality to the perfusion chromatography.
(3) Taking inorganic particles as pore-making agent: Y. Sun et al prepared bulk-shaped polymers with a pore diameter of about 340 nm with a double pore-making agent method using solid particles and solvents, further ground them into sphere-shaped particles, and extended this method to sphere-shaped media (see Y. H. Yu, Y. Sun. Macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) matrix as an anion-exchange resin for protein adsorption, Journal of Chromatography A, 1999, 855: 129-136; M. L. Zhang, Y. Sun. Poly(glycidyl methacrylate-divinylbenzene-triallylisocyanurate) continuous-bed protein chromatography, Chromatography A, 2001, 912: 31-38; M. L. Zhang, Y. Sun. Coorperation of solid granule and solvent as porogenic agents novel porogenic mode of bioporous media for protein chromatography, Chromatography A, 2001, 922: 77-86; Y. Sun, M. L. Zhang, M. Bai et al. Chinese Patent Application No. 01118231.8). These super-macroporous media attained good results in separation application. They further found that, in order to obtain the through large pore diameters, the volume content of the inorganic particles should be 10-40%. However, though a high embedding ratio can be attained by embedding the hydrophilic inorganic particles into the hydrophilic agarose, the compatibility between most synthesized macromolecule materials (such as polystyrene and the like) and inorganic particles is poor. Therefore, it is difficult to embed a large amount of the inorganic particles into these materials completely.
(4) PolyHIPE, high internal phase emulsion polymerization: it was published in 1985 by Barby et al (see U.S. Pat. No. 4,522,953), wherein a great amount of water was dispersed into a monomer to prepare a W/O type emulsion. Because the volume of the internal aqueous phase exceeded 70%, it was referred as high internal phase emulsion (HIPE). A Bulk-shaped macroporous material was obtained by subjecting this HIPE to polymerization. Because the volume of the internal phases is high and the aqueous phases will be penetrated mutually after the polymerization, mutually penetrated macropores will be formed after drying. At the beginning of 1990s, a new super-macroporous sphere-shaped medium prepared based on the high internal phase emulsion polymer technology, that is, the so-called Magnapore Material, was disclosed in U.S. Pat. No. 5,583,162, U.S. Pat. No. 5,653,922, U.S. Pat. No. 5,863,957, U.S. Pat. No. 6,100,306, Chinese Patent Application No. 95193484.8 by Naihong Li et al. This kind of microsphere is characterized in that, the channels are regular, the pore diameters are relatively large, and the interconnected pores are obvious. This microsphere has a diameter of 50-300 μm, a pore diameter of 1-50 μm, a density of 0.05-0.2 g/cm2, a porosity of 70-90% and a specific surface area of 2-30 m2/g. The preparation method thereof is as follows: firstly, dispersing the internal aqueous phase into an oil phase to form a high internal phase emulsion (the internal aqueous phase has a volume exceeding 70%); then dispersing the high internal phase emulsion into an external aqueous phase to form a W/O/W emulsion; and carrying out a polymerization. Because the aqueous phase in the high internal phase emulsion has high volume content and poor stability, many factors, such as, the hydrophobicity/hydrophilicity and volume fraction of the monomers in oil phase, the kind and concentration of the surfactants, the viscosity of the continuous phase and the polymerization temperature, will affect the high internal phase emulsion. If the control is inappropriate, it is easy to cause emulsion breaking which results in that it is impossible to form microspheres.
These methods in the prior art all have the disadvantages of troublesome preparation method, complex formulation, poor controllability and inapplicability in large scale production. Additionally, the polymeric microspheres prepared by these methods are inadequate in mechanical strength and may be broken partially in actual application, which will affect the separation effect thereof.